Explanation

Methomyl (S-methyl-N-[(methylcaramoyl)oxy]thioacetamidate) was evaluated toxicologically by the JMPR in 1978, 1986 and 1989 (Annex 1, references 30, 47 and 56). In 1989, an ADI of 00.03 mg/kg bw was established on the basis of a NOAEL of 3 mg/kg bw per day in a 2-year study of toxicity in dogs and a 100-fold safety factor. This ADI was maintained by a 1994 WHO Core Assessment Group that prepared Environmental Health Criteria 178 (WHO, 1996). The Meeting was asked to establish an acute reference dose (RfD) for methomyl by the Codex Committee on Pesticide Residues. The present Meeting therefore evaluated data submitted in support of an acute RfD and additional data on the toxicity of repeated doses and of dermal application and on genotoxicity.

All the studies submitted complied with the essential elements of the applicable test guidelines, except where noted specifically in the text.

Evaluation for acceptable daily intake

1. Toxicological studies

(a) Short-term studies of toxicity

Rabbits

Methomyl (purity, 98.4%) was applied to the clipped intact skin of groups of New Zealand white rabbits of each sex at a dose of 0 (10 animals), 5, 50 (five animals) or 500 (10 animals) mg/kg bw per day for 21 days. The doses were applied in water each day, and the site was occluded for 6 h with a non-porous, plastic dressing. Half the males and females given 0 or 500 mg/kg bw per day and all animals in the other groups were killed at the end of dosing. The remaining animals at 0 or 500 mg/kg bw per day were allowed to recover for 14 days and were then killed. Blood samples for measurement of cholinesterase activity were taken after the final exposure, stored on ice and assayed as quickly as possible (usually with 30 min). Brain samples for determination of cholinesterase activity were stored at 70 ΊC until analysis.

The only clinical change was an increased incidence of hyperactivity at 500 mg/kg bw per day. There were no effects on body-weight gain, haematological or biochemical end-points, organ weights or organ histology. Plasma cholinesterase activity was reduced to 36% and 55% of the control mean at day 21 in males and females at the highest dose, respectively. Brain cholinesterase activity was depressed to 48% and 68%, respectively, in males and females at this dose, but erythrocyte cholinesterase activity was not decreased to a biologically significant extent. In males at 50 mg/kg bw per day, plasma cholinesterase activity was depressed to 77%, although most individual values were within the range of the individual control values, and there was no inhibition of erythrocyte cholinesterase activity in animals of either sex. All the values for cholinesterase activity had returned to control values by the end of the 14-day recovery period. The NOAEL was 50 mg/kg bw per day, on the basis of significant inhibition of brain acetylcholinesterase activity at 500 mg/kg bw per day (Brock, 1989).

Methomyl (purity, 98.6%) was applied to the clipped intact skin of groups of six male and six female New Zealand white rabbits at a dose of 0, 15, 30, 45 or 90 mg/kg bw per day for 21 days. The material was applied in water for 6 h, and the area was covered with a porous, non-occlusive wrapping. The study was intended to define more accurately the NOAEL in the earlier study (Brock, 1989). Standard assessments were made, including observations for deaths and clinical signs of toxicity and measurements of body weight and food consumption. One hour after the final exposure, blood samples were collected for measurement of erythrocyte and plasma cholinesterase activity, after which the animals were killed. Gross necropsy was performed, and the brains were collected and stored frozen for determination of cholinesterase activity.

No deaths occurred during the study, and no significant differences in mean body weight, mean body-weight gain, food consumption or food efficiency were noted. No significant signs of toxicity attributable to exposure were seen. No gross lesions were found at necropsy in any treated animals. Slight but statistically significant reductions in mean brain cholinesterase activity were seen in males at doses > 30 mg/kg bw per day, although there was no clear doseresponse relationship: the mean values were 99, 90, 88 and 90% of the control value at 15, 30, 45 and 90 mg/kg bw per day. There was some evidence of a pattern in the individual values for males, which were below the control range in 1/6, 3/6, 5/6, 4/6 males at 15, 30, 45 and 90 mg/kg bw per day, respectively. In females, a statistically significant reduction in brain cholinesterase activity was found at the highest dose (94% of the control value). Overall, these reductions in males and females were not biologically significant. There was no statistically significant effect on erythrocyte cholinesterase activity in animals of either sex, although the group mean erythrocyte cholinesterase activities were lower in all treated groups apart from females at 30 mg/kg bw per day; the group mean values for males were 7087% of the control value and those for females were 88103%. Comparison of individual values indicated that they were within the range of biological variation. The plasma cholinesterase activities in the treated groups were also generally lower than in controls, although the changes were not statistically significant, nor was there evidence of a doseresponse relationship in either group means or individual values. The NOAEL for dermal toxicity was 90 mg/kg bw per day (Finlay, 1997).

(b) Genotoxicity

The results of two newly submitted assays for genotoxicity are shown in Table 1. Methomyl was not genotoxic in vitro or in vivo.

Table 1. Results of tests for genotoxicity with methomyl

End point

Test object

Concentration

Purity
(%)

Results

Reference

In vitro

Reverse mutation

S. typhimurium

TA97a, TA98, TA100, TA1535, E. coli WP2 uvrA (pKM101)

105000 ΅g/plate in dimethyl sulfoxide

98.4

Negative with and without metabolic activation

Mathison (1997)

In vivo

Micronucleus formation

CD-1 mice

312 mg/kg bw in water

98.4

Negativea

Bentley (1995)

a Single treatment-related death at 12 mg/kg bw

(c) Special studies

(i) Reversibility

The time necessary for recovery from the inhibition of cholinesterase activity caused by oral administration of methomyl (purity, 98.4%) was investigated in groups of 40 adult Crl:CD BR Sprague-Dawley rats of each sex given methomyl in deionized water once by gavage at a dose of 0 or 3 mg/kg bw. Blood was collected from 10 animals of each sex per group 30 min, 2, 3 and 4 h after dosing. Immediately after blood collection, the rats were killed and their brains removed and frozen for subsequent analysis of cholinesterase activity. The sampling times were based on pilot studies that indicated a time-to-peak effect of 0.5 h for both plasma and erythrocyte cholinesterase activity over a dose range of 315 mg/kg bw. Clinical signs of toxicity were assessed before collection of blood at each time.

Tremors were seen in 17/40 males and 6/40 females treated with methomyl within 30 min of dosing, but no clinical signs were seen later. The treated animals showed marked, statistically significant inhibition of cholinesterase activity 30 min after treatment, the group mean values being 73% of the control mean for plasma, 44% for erythrocytes and 54% for brain in males, and 90, 59 and 61% of the control means in females, respectively. Two hours after treatment, the males showed statistically significant inhibition of blood and brain cholinesterase activities, the group means being 81% of the control mean for plasma, 76% for erythrocytes and 84% for brain. After 3 h, only brain cholinesterase activity in males showed statistically significant inhibition (92% of control value); however, after 4 h, the brain activity was similar to that of controls. In females 2, 3 and 4 h after treatment, the blood cholinesterase activity was not significantly different from that of controls. At 2 and 3 h, there was a minimal but statistically significant reduction in brain cholinesterase activity (92% of control value at both times) (Malley, 1997).

(ii) Neurotoxicity

Groups of 52 Crl:CD BR Sprague-Dawley rats of each sex received methomyl (purity, 98.6%) by gavage at a dose of 0, 0.25, 0.5, 0.75 or 2 mg/kg bw in an aqueous solution. Twelve rats from each group were used to evaluate neurotoxicity, and clinical observations, body weights and food consumption were recorded periodically. A functional observational battery (FOB) and motor activity tests were administered before treatment, about 30 min after dosing on day 1 and again on days 8 and 15. Six rats of each sex per group for evaluation of neurotoxicity were killed, and their tissues were fixed in situ by perfusion on day 16. Tissues from the control group and those at the highest dose were processed and examined for neuropathological effects. Forty rats of each sex per group were used to evaluate clinical pathology. Cholinesterase activity in plasma and erythrocytes was measured in the first 10 rats per group before treatment, in order to establish baseline levels. Cholinesterase activity in brain, plasma and erythrocytes was measured again in the same 10 animals per group about 30 min after dosing on day 1 and in another 10 animals per group on day 2. The remaining 20 animals per group used to evaluate clinical pathology were evaluated on days 1 and 2; however, as they were not needed for measurement of cholinesterase activity on days 8 and 15, they were removed from the study. Brain samples were kept at  70 °C until analysis.

Body-weight gain was decreased in females at 2 mg/kg bw during days 28 but returned to control values during days 815. Administration of the FOB on day 1 (30 min after dosing) showed tremors and lachrymation in male rats at 2 mg/kg bw, but no treatment-related effects were seen on days 8 and 15. Although tremors also occurred in females, they occurred in one or two animals in all groups, including controls, indicating no relationship to treatment. There were no effects on forelimb grip strength, hindlimb grip strength or hindlimb foot splay. The motor activity scores of treated rats were unaffected. Clinical signs of toxicity were observed in animals at 2 mg/kg bw, which included tremors (both sexes), salivation (females) and/or lachrymation (both sexes) about 30 min after dosing. No clinical signs of toxicity were observed on day 2.

Inhibition of blood and brain cholinesterase activity was detected in males and females at doses > 0.5 mg/kg bw (Table 2) on day 1 about 30 min after treatment. Cholinesterase activity was comparable to that of controls on day 2. Neuropathological evaluation revealed no treatment-related adverse findings. The NOAEL was 0.25 mg/kg bw on the basis of statistically significant, > 20% inhibition of brain and erythrocyte acetylcholinesterase activity at doses > 0.5 mg/kg bw (Mikles, 1998a).

Table 2. Inhibition of cholinesterase activity (% of control) in rats 30 min after a single dose of methomyl by gavage

Tissue

Dose (mg/kg bw per day)

Males

Females

0.25

0.5

0.75

2.0

0.25

0.5

0.75

2.0

Plasma

83

87

77*

58*

89

77

67*

72*

Erythrocytes

102

95

70

54*

85

75*

62*

43*

Brain

94

81*

75*

53*

96

80*

70*

49*

From Mikles (1998a)

* p < 0.05

Groups of 10 male Crl:CD BR Sprague-Dawley rats were given 10 g of feed containing methomyl (purity, 98.4%) at a concentration of 0, 30, 60, 120 or 360 ppm, equal to 1, 1.9, 3.7 and 10 mg/kg bw. The animals were conditioned to eat within 2 h. During treatment, the animals were observed for clinical signs of toxicity. About 1 h after the end of feeding, each rat underwent an FOB that followed the guidelines of the USA's Environmental Protection Agency, except that grip strength and foot splay were not assessed quantitatively, and the evaluators were aware of which animals had been treated. When the FOB was completed, blood was collected for determination of plasma and erythrocyte cholinesterase activity. The rats were subsequently killed, and brain cholinesterase activity was determined. The mean interval between blood and brain sampling and the start of treatment was 3 h, and the interval before the end of treatment was 11.5 h. Blood and brain samples were kept on ice until analysis.

No signs of toxicity were observed during treatment. During the FOB, a pattern of changes was seen at concentrations > 60 ppm but was most apparent at 360 ppm. These changes consisted of increased incidences of low arousal, no reaction to approach or touch stimulus and no reaction to tail pinch, although only the last showed a clear dose-related trend at concentrations > 60 ppm. Statistically significant reductions (> 20%) in erythrocyte and brain cholinesterase activity were seen at concentrations > 120 ppm. Plasma cholinesterase activity was also significantly reduced at 120 ppm (by 17%) and 360 ppm (by 37%). However, the individual values were variable, particularly for erythrocyte cholinesterase activity, and there was a poor correlation between the three end-points. The NOAEL was 30 ppm, equal to 1 mg/kg bw, on the basis of the increased incidence of diminished response to tail-pinch at 60 ppm (Filliben, 1999).

Groups of 42 Crl:CD BR Sprague-Dawley rats of each sex received diets containing methomyl (purity, 98.6%) at a concentration of 0, 20, 50, 150 or 1500 ppm for about 13 weeks, equal to 0, 1.3, 3.1, 9.4 and 95 mg/kg bw per day for males and 0, 1.5, 3.9, 11 and 110 mg/kg bw per day for females. All animals were observed for clinical changes, body weights and food consumption weekly throughout the study. Twelve rats of each sex per group were evaluated for neurobehavioural changes in an FOB and a motor activity test before treatment (baseline) and again during weeks 4, 8 and 13. Six rats of each sex per group was perfused in situ after week 13. Tissues from all rats were saved, but only those from controls and rats at 1500 ppm were evaluated microscopically. Thirty rats of each sex per group were used to evaluate clinical pathology. Cholinesterase activity in plasma and erythrocytes was measured in the first set of 10 rats of each sex per group before treatment to establish baseline levels. The cholinesterase activities in brain, plasma and erythrocytes were measured again in the same rats during week 4, in the second set during week 8 and in the third set during week 13. Blood samples were kept on ice and brain samples were kept at 70 °C until analysis. The study was possibly compromised, as the animals were fasted before sampling, the duration of food withdrawal not being stated.

No treatment-related deaths occurred during the study. Animals of each sex at 1500 ppm showed tremors during the early part of the study, and males in this group also showed increased incidences of aggressive behaviour and hyperreactivity. An increased incidence of alopecia was observed in females in this group. Statistically significant decreases in body weight, body-weight gain, food consumption and food efficiency were seen in male and female rats at 1500 ppm.

During the FOB, the animals at 1500 ppm had increased incidences of difficult removal from their home cages, difficult handling, ptosis and abnormal pupillary responses (more dilated than normal for lighting conditions, slow and incomplete reactions to light). The females also had decreased defaecation and urination and increased incidences of low arousal and abnormal gait. Statistically significant decreases in forelimb and hindlimb grip strength were seen in males at 1500 ppm in week 13. Neuropathological evaluation revealed no findings related to treatment. Brain cholinesterase activity was reduced in males and females at 1500 ppm, which was statistically significant only at week 8 in males (81% of control) and at week 4 in females (90% of control). The reductions at other sampling times were minimal and not statistically significant. There was no evidence of inhibition of erythrocyte cholinesterase activity in any group, although there was considerable variation (without any obvious pattern) between group means and over time. There was some evidence of mild inhibition of plasma cholinesterase activity in males at 1500 ppm during assessment at week 8 (92% of control).

2. Observations in humans

In a randomized double-blind study with ascending doses, groups of five healthy male volunteers (four controls) aged 1840 years received single oral doses of a methomyl formulation. containing 89% methomyl or a placebo (the inert ingredient of the formulation, hydrated silica) in a capsule at a dose of 0, 0.1, 0.2 or 0.3 mg/kg bw. All volunteers were informed of the nature of the test substance, and written informed consent was obtained. The protocol and information were reviewed and agreed by an independent ethics committee. The doses were administered 5 min after a standard breakfast. The men were observed for two nights and attended a follow-up visit 7 (± 2) days after dosing. Blood samples for analysis of cholinesterase activity were taken 16 h (admission) and 0.5 h before dosing and 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 12 and 24 h and 7 days (± 2) after dosing, separated immediately and chilled in liquid nitrogen.

No adverse events requiring treatment with atropine were observed during the study. There were no treatment-related effects on the electrocardiogram, heart rate, pulse, blood pressure, respiratory rate, body temperature, haematological or clinical chemical parameters (excluding cholinesterase activities), urinary end-points or the pupil. The only treatment-related effects were statistically significant decreases in erythrocyte (Table 3) and plasma cholinesterase activity, a single occurrence of mild headache and quantitatively increased salivation, detected by measuring the mass of secretion (Table 4). The biological significance of the increase in salivary secretion is questionable, as, although the men secreted more saliva than before treatment, the mass of secretion was similar to that of controls.

Table 3. Inhibition of erythrocyte cholinesterase activity (% change from baseline)a in male volunteers after a single oral dose of methomyl in a capsule

Table 4. Saliva production (g/5 min) in male volunteers receiving a single dose of methomyl

Dose

(mg/kg bw)

30 min before dosing

At time of peak effect after dosing

Mean

Max

Mean

Max

0

2.6

4.1

2.6 (4 h)

4.2

0.1

0.9

2.1

1.5 (6 h)

4.1

0.2

1.3

3.0

2.3 (8 h)

5.2

0.3

1.4

2.1

2.5 (1 h)

3.6

From McFarlane et al. (1998)

Men at 0.1 mg/kg bw showed a notable decrease (19% below baseline value) in group mean erythrocyte cholinesterase activity 1.25 h after dosing (p = 0.072, Student t test), two members of the group having values about 30% lower than their baseline values. However, there were no clear indications of a treatment-related pattern at other times or in the plasma cholinesterase activity in these or other individuals at this dose. Men at 0.2 mg/kg bw had a statistically significant increase in measured salivary secretion 3 h after dosing when outliers were excluded from the data set. Statistically significant depressions of erythrocyte and plasma cholinesterase activities were seen 45 min and 2 h after dosing, but both activities had returned to baseline level within 6 h. Men at 0.3 mg/kg bw showed a statistically significant change from baseline in measured salivary secretion 1 h after treatment when outliers were excluded from the data set. Both erythrocyte and plasma cholinesterase activities were statistically significantly inhibited 15 min to 4 h after dosing. Both activities had returned to baseline levels within 6 h. One man reported mild headache about 1 h after maximal erythrocyte cholinesterase inhibition.

Comments

The acute LD50 of methomyl administered orally was approximately 20 mg/kg bw in rats. The compound is classified by WHO (1999) as highly hazardous.

Methomyl was tested for genotoxicity in a range of studies in vitro and showed cytogenetic potential in one study in human lymphocytes; it was not cytogenetic in rats in vivo. In studies evaluated by the present Meeting, methomyl did not induce gene mutation in vitro or micronuclei in mice treated in vivo. The Meeting concluded that methomyl is unlikely to be genotoxic.

Methomyl is not a reproductive or a developmental toxicant.

Rats given methomyl by gavage at a dose of 3 mg/kg bw showed peak neurotoxic effects (clinical signs and inhibition of erythrocyte cholinesterase activity) at 30 min and almost complete recovery by 2 h. In a study with single doses, male rats conditioned to eat within 2 h received methomyl at a single dose of 0, 1, 1.9, 3.7 or 10 mg/kg bw. Significant reductions (> 20%) in erythrocyte and brain cholinesterase activities were seen at doses > 3.7 mg/kg bw. The NOAEL was 1 mg/kg bw on the basis of a dose-related diminution in response to tail pinch at doses > 1.9 mg/kg bw. In another study, rats received methomyl at a dose of 0, 0.25, 0.5, 0.75 or 2 mg/kg bw by gavage in an aqueous vehicle. The NOAEL was 0.25 mg/kg bw on the basis of rapidly reversible inhibition of erythrocyte and brain cholinesterase activity.

In a 13-week study of neurotoxicity, groups of rats received diets containing methomyl at a concentration of 0, 20, 50, 150 or 1500 ppm. At 1500 ppm (equal to 95 mg/kg bw per day), several treatment-related effects were seen in animals of each sex, including decreased brain cholinesterase activity, tremors and abnormal pupil responses. The NOAEL was 150 ppm, equal to 9.4 mg/kg bw per day. The Meeting noted that this NOAEL observed after repeated dietary administration was higher than the NOAELs observed in the studies with single doses described above.

Male volunteers received single capsules containing methomyl at a dose of 0, 0.1, 0.2 or 0.3 mg/kg bw soon after breakfast. On the basis of dose-related, statistically significant inhibition of erythrocyte cholinesterase activity, by > 20%, and a statistically significantly increase in saliva secretion at doses > 0.2 mg/kg bw, the NOAEL was 0.1 mg/kg bw.

Toxicological evaluation

The Meeting allocated an acute RfD of 0.02 mg/kg bw on the basis of the NOAEL of 0.1 mg/kg bw in the study with volunteers. A safety factor of 5 was used because the effects were rapidly reversible and driven by the maximal concentration in plasma (see Annex 1, reference 91, Annex 5). This value was supported by the results of the study of acute neurotoxicity in rats treated in the diet, with a NOAEL of 1 mg/kg bw, the study of acute neurotoxicity in rats treated by gavage, with a NOAEL at 0.25 mg/kg bw, and the absence of any significant sex difference in studies in rats.

The Meeting noted that this acute RfD was lower than the current ADI. This is plausible in view of the toxicological profile of methomyl, which shows very rapid recovery from cholinesterase inhibition, such that the NOAELs for dietary intake over 2 h or over 13 weeks were higher than the NOAEL for a single bolus dose. For this reason, setting an acute RfD on the basis of a single meal rather than daily consumption might be justified. Practical implications associated with this situation were noted, as the data on intake do not allow subdivision of daily intake into individual meals. The Meeting concluded that the ADI and acute RfD for methomyl should be based on the same NOAEL and revised the ADI to 00.02 mg/kg bw on the basis of the NOAEL of 0.1 mg/kg bw in the study with volunteers and a safety factor of 5.